Fluorescent dyes are widely used in biological research and medical diagnostics. Fluorescent dyes are superior to conventional radioactive materials because fluorescent dyes are less expensive and less toxic, and can typically be detected with sufficient sensitivity. In particular, a diversity of fluorophores with a distinguishable color range has made it more practical to perform multiplexed assays capable of detecting multiple biological targets at the same time. The ability to visualize multiple targets in parallel is often required for delineating the spatial and temporal relationships amongst different biological targets in vitro and in vivo. In addition, the generation of a wide range of fluorescent dyes has opened a new avenue for conducting high-throughput and automated assays, thus dramatically reducing the unit cost per assay.
Fluorescent nuclear stains are fluorescent dyes that can selectively stain the nuclei of cells. Nuclear stains may be divided into those that can cross cell membranes and those that cannot. Membrane permeable nuclear stains are useful for reveal the natural location of live cells and also for monitoring the nuclear changes during cell processes such as meiosis, mitosis and apoptosis. On the other hand, cell impermeant nuclear stains can be used to mark the nuclei of fixed cells or to selectively detect dead cells in the presence of live cells. Nuclear counterstains are particularly useful for immunofluorescence studies, where being able to fluorescently mark the nucleus of every single cell provides a means to analyze the immunostaining of sub-populations of cells relative to the entire cell population.
A number of fluorescent nuclear counterstains are commercially available. Examples of widely used nuclear counterstains include DAPI, Hoechst 33258, Hoechst 33342 and propidium iodide (PI) (Kenneth, H. J. and Douglas, A. K., Journal of Histochemistry and Cytochemistry 35, 123 (1987)). DAPI and the Hoechst dyes are UV-excitable blue fluorescent nucleic acid dyes. A major drawback of UV-excitable dyes is that relatively few confocal microscopes and other laser-based imaging instruments are equipped with UV excitation source. In addition, DAPI and the Hoechst dyes tend to have broad emission spectra that extend the fluorescent signal into the green and yellow detection channels, interfering with the detection of other fluorescent probes. PI has an absorption peak at ˜530 nm and emission at ˜610 nm. However, the 530 nm absorption peak of PI does not optimally match with the most common laser excitation wavelengths available. Furthermore, the 610 nm emission wavelength of PI is still not sufficiently long enough to separate from some of the orange and red fluorescent dyes widely used in immunofluorescence and other cellular staining. Various SYTO dyes from Invitrogen are available as nuclear counterstains for live cells (Bkaily et al., Mol. Cell. Biochem. 172, 171 (1997)). SYTO dyes have fluorescence wavelengths that occupy a spectral region otherwise more valuable to be reserved for other fluorescent probes in multiparameter imaging or detection. TOTO and YOYO dyes are membrane-impermeant nuclear stains. These are dimeric cyanine dyes carrying multiple positive charges for enhanced DNA-binding affinity. TOTO and YOYO dyes show high cytoplasmic background due to staining to RNA. Draq5 and Draq7, two DNA-binding dyes developed by Biostatus (U.S. Pat. Nos. 7,605,280; 7,060,427; and 6,468,753), are nuclear counterstains with far-red to near infrared fluorescence emission, which does not interfere with other detection channels in the visible spectral region useful for other probes. See also US Patent Application 2010/0062429. However, for fixed cells, the nuclear specificity of both Drq5 and Draq7 suffers, which poses problems for their application in immunofluorescence staining, where cell fixation and permeabilization is routine practice.
Thus, there remains a need for alternate fluorescent nuclear stains.